Research objectives are directed toward understanding the basis for the recognition and processing of radiation induced lesions in known radiation hit and known non-hit bystander cells. Nuclear DNA changes significantly impact human health with clear relationships to cell death, mutation and oncogenic change. The basic paradigm that the directly damaged cell nucleus is the only important radiation target has been brought into question. This questioning arouses significant uncertainties about the basis for extrapolation of hazard to human radiation exposure in the low dose region, and also impacts on appropriate target volume in radiation therapy. An understanding of the role of the fraction of hit cells in a population and of sub-cellular components in radiation responses can efficiently be obtained by use of a microbeam. A directed beam of high LET charged particles can initiate significant levels of damage in sub-cellular micro-volumes. Single cell microscopic examination can localize gene product/s to sub-cellular regions. Single cell gene expression analyses of known hit and known bystander cells complement this approach. In this proposal we will use recently established protocols for cell site specific microbeam irradiation to undertake a systematic evaluation of the role of intercellular distance, including cell contact, in both human fibroblast and human epithelial cell populations, individually and mixed. It is hypothesized that gap junction intercellular communication is a minor component of the bystander effect.